Epigenomic heterogeneity and spatial organization during human reprogramming

Reprogramming somatic cells to pluripotency requires extensive remodeling of transcriptional and epigenetic states, yet most cells never complete the transition. We integrated single-cell gene expression, chromatin accessibility, DNA methylation and three-dimensional genome organization across densely sampled reprogramming time courses of fibroblasts from four individuals, together with highly multiplexed spatial transcriptomics.

1. 1DNA methylation carries individual identity that other layers miss. Starting fibroblasts show extensive inter- and intra-individual methylation heterogeneity — concentrated in partially methylated domains — that is far less apparent in RNA, ATAC or 3D contacts, and diminishes only along the productive trajectory.
2. 2The two failure branches fail differently. Cells diverging early acquire inflammatory and antiviral transcription while their methylation and 3D-genome changes revert toward the fibroblast state. Cells that stall later advance transcriptionally but leave methylation remodeling incomplete.
3. 3An early-iPSC transcriptome is not one endpoint. Cells reaching a similar early iPSC transcriptional state separate into two distinct methylation and 3D-genome configurations, one far closer to mature iPSCs — with 80.9% of differentially methylated regions overlapping differential TAD boundaries.
4. 4Fate is spatially organized by day 3. In situ profiling of 139,407 cells shows productive cells already concentrated in hotspots at the earliest sampled stage, with fewer hotspots in the low-efficiency donor.